Mir 223 3p

Nine miRNA were chosen for RT-qPCR validation; miR-29a-3p (TaqMan® ID 007600_mat, Applied Biosystems), miR-99a-5p (TaqMan® ID 006254_mat), miR-126-3p (TaqMan® ID 008451_mat), miR-140-3p (TaqMan® ID 471823_mat), miR-222-3p (TaqMan® ID 000525), miR-223-3p (TaqMan® ID 002295), miR-204-5p (TaqMan® ID 000508), miR-409-3p (TaqMan® ID 002332), miR-6119-5p (Custom TaqMan® small RNA Assay). Reverse transcription was achieved on 10 ng of total RNA using the TaqMan® MicroRNA Reverse Transcription (Applied Biosystems, Foster City, CA, USA) kit following the manufacturer’s instructions. In the thermal cycler (StepOne+, Applied Biosystems, Foster City, CA, USA), each 15 μL RT reaction followed 30 min at 16°C, 30 min at 42°C, 5 min at 85°C. Then, 1.3 μL of miRNA-specific cDNA from the reaction were amplified using the TaqMan® Small RNA Assays (Applied Biosystems, Foster City, CA, USA) following the manufacturer’s instructions. Amplification was performed at 95°C for 10 min, pursued by 40 cycles of 95°C for 15 s and 60°C for 1 min. All miRNA levels were normalized to the values of U6 snoRNA [47 (link),48 (link)].

miRNAs were determined using two-step RT-qPCR with RT-primer specific assay in combination with TaqMan probes: miR-143-5p (Assay ID: 463509_mat), miR-140-5p (Assay ID: 001187) miR-148a-5p (Assay ID: 473012_mat), miR-450a-5p (Assay ID: 462729_mat), miR-21-5p (Assay ID: 000397), miR-146b-5p (Assay ID: 002755), miR-342-3p (Assay ID: 002260), and miR-223-3p (Assay ID: 07896_mat) (Applied Biosystems; CA, USA). Each RT-reaction used 1.5 µL from the 14 µL eluted RNA using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems; CA, USA). The RT reaction program and PCR cycling conditions were as we previously reported (Brianza-Padilla et al., 2016 (link)). miRNAs relative concentrations were normalized with Ct values of cel-miR-39, and values were calculated using 2^−ΔΔCt and 2^−ΔCt formulas. All Ct values for cel-miR-39 ranged from 20 to 22 cycles both for total plasma and for EVs RNA isolations.